One of the most devastating diseases to manage is metastatic melanoma, where the evidence of stem cell subpopulations is starting to emerge. Comparative global gene analyses of aggressive human melanoma cell lines and tumors have revealed the expression of genes associated with multiple cellular phenotypes and precursor stem cells. These findings support the premise that aggressive melanoma cells revert to a multipotent, plastic phenotype. Key to identifying the molecular mechanisms underlying tumor cell plasticity is to elucidate the unique role the microenvironment plays in this process. Most noteworthy are the data we have generated showing the reprogramming of subpopulations (possibly the stem cells) of multipotent metastatic melanoma cells to a melanocyte-like phenotype by the embryonic microenvironment(s) of hESCs and chick neural crest-rich regions. Based on these observations, we propose to test the central hypothesis that the embryonic microenvironments associated with hESCs (Wa01. Wa09 and UC06) and chick neural crest-rich region(s) - contain informational cues with the potential to epigenetically reprogram the genotype and phenotype of human metastatic multipotent melanoma cells exposed to them. Our long term goal is to understand the biological mechanisms underlying the bi-directional communication between stem cells and their microenvironment(s) that ultimately result in cell fate determinations. Our short term goal is to identify the biological and molecular parameters associated with the epigenetic reprogramming of multipotent metastatic melanoma cells exposed to specific hESC environmental cues. Using unique, 3-D organotypic models and chick embryos, together with functional analysis, 4-D imaging, laser capture microdissection, and epigenetic analysis, we propose to: Unchanged Aim 1: Determine the epigenetic influence of the 3-D microenvironment(s) of hESCs for their potential to reprogram the genotype and phenotype of human metastatic multipotent melanoma cells. Revised Aim 2: Identify the molecular basis for the epigenetic reprogramming of the genotype and phenotype of the affected multipotent melanoma cell populations exposed to various hESC 3-D microenvironments, with particular focus on the Nodal signaling pathway. Revised Aim 3: Investigate the developmental plasticity of multipotent melanoma cell populations in an embryonic chick model - to determine the biological relevance of the Nodal signaling pathway involved in stem cell plasticity and the control of cell fate determination and reprogramming of the metastatic phenotype. Lay Summary: At the completion of these studies, we expect to gain new insights into the biological properties of human embryonic and multipotent metastatic melanoma cells with stem cell properties that could be translated for novel therapeutic applications.